Expression Of Autophagy Related Molecule And Mechanism Of Ca-A/K Channel In Hypoxic-ischemic Model Of Neonatal Rats’ Hippocampus In Vitro

Objective: To study the injury of hippocampal neuron, the changes of autopagy and subunit of Ca-A/K channel after injury in different points in time and groups, to discuss the effect of 3-methyladenine(3-MA) on regulation of autophagy and cell survival, and to have a preliminary study on relationship of sununit of Ca-A/K channel and autophagy.Method: The neuron hypoxic-ischemic model was made by oxygen glucose deprivation(OGD) in primary cultured hippocampal neuron from neonatal Sprague Dawley rat within 24 hours after birth. Western blot and RT-q PCR was used to detect the changes of hippocampal neuron autophagy related protein(LC-3, Beclin-1), m RNA, and subunit of Ca-A/K channel(Glu R1, Glu R2, Glu R3) at 0 h, 6h, 12 h, 24 h, 48 h, 72 h and 96 h after OGD. Specific structure of autophagy was detected by transmission electron microscope,cell activity by CCK8; the changes of [Ca2+]i by streaming Fluo-3 AM; then preliminary discussed the relation and mechanism of autophagy and Ca-A/K Channel.Result:1. Electron microscope showed that more autophagosome formed in neuron after hypoxia and ischemia than in normal condition; and autophagosome also formed in 3-MA intervened cells, but was significant less than hypoxic-ischemic group.2. RT-q PCR showed that expression of LC-3 and Beclin-1 elevated in hypoxic-ischemic group(0.5h and 1h) compared with controlled group, they elevated at 0h in hypoxic-ischemic group(0.5h), and reached peak level at 48 h, then decreased gradually,while peak level at 72 h then decreased in hypoxic-ischemic group(1h), and was higher than group(0.5h); LC-3 and Beclin-1 decreased at 6h and reached normal level at 12 h in 3-MA intervened group.3. Western blot showed that expression of LC-3 and Beclin-1 elevated in hypoxic-ischemic group(0.5h and 1h) compared with controlled group, they reached to peak level at 6h in hypoxic-ischemic group(0.5h), and decreased after 48 h, while peak level at 12 h then decreased in hypoxic-ischemic group(1h), and was higher thanhypoxic-ischemic group(0.5h); LC-3 and Beclin-1 showed a downward trend at 6h in3-MA intervened group. Expression of Glu R1 and Glu R3 elevated at 6h after hypoxia and ischemia in hypoxic-ischemic group(1h), and began to decrease at 24 h, while Glu R3 expression decreased at 6h, and recovered at 24 h.4. CCK8 showed that for hypoxic-ischemic injury, 24 h cell survival rate was 71±1%, and 48 h survival rate was 80±4%%, and the survival rate in 3-MA intervened group were 75±5% and 82±4%%, and there was no significant difference(p>0.05).Conclusion:1. We succeed in culturing the primary hippocampal neuron, and build hypoxic-ischemic model.2. Autophagy marker protein LC-3, Beclin-1 and subunit of Ca-A/K channel Glu R1,Glu R2, Glu R3 change after hypoxia and ischemia.3. In limited injury, autophagy increases as time goes after hypoxia and ischemia.4. Autophagy inhibitor 3-MA can impact on the expression of autophagy related protein, and decrease cell injury.5. Structure of Ca-A/K channel change after hypoxia and ischemia, suggests the formation of a new channel which subunit Glu R2 decreases, it can mediate Ca2+ influx, and induce the autophagy.